No large-scale immunomodulatory clinical therapies currently exist for heart failure (HF). To date, the primary focus of therapeutic immunomodulation has been to neutralize inflammatory mediators such as tumor necrosis factor-?, an approach that unfortunately failed in clinical trial. Recently, we described profound remodeling of the global immune cell network and a heretofore-unappreciated adverse cardiosplenic axis in chronic ischemic HF that promotes immune cell-mediated tissue injury in the failing heart. This novel discovery suggests that directly targeting specific leukocyte populations that are altered in HF may represent a better approach to therapeutic immunomodulation. Regulatory T-cells (Tregs) are CD4+ T-lymphocytes that express the forkhead box protein-3 (Foxp3) transcription factor and suppress immune cell responses in a contactdependent, antigen nonspecific manner; their pathophysiological role in chronic HF is unknown. In our preliminary studies, we have unexpectedly discovered that in HF, Tregs robustly infiltrate the failing heart, and rather than being anti-inflammatory and thereby cardioprotective, paradoxically exhibit pro-inflammatory features and function prominently as disease mediators, imparting substantial anti-angiogenic, pro-fibrotic, and pro-inflammatory effects. Based on these data, we hypothesize that dysfunctional Tregs are essential for left ventricular (LV) remodeling, capillary rarefaction, inflammation, and disease progression in HF, and thereby are key cellular targets for immunomodulation. Three Aims will test this hypothesis. In Aim 1, we will delineate global Treg trafficking and alterations in Treg pro-inflammatory signaling in C57BL/6 mice with coronary ligation and HF, and in sham-operated controls. Tregs, and other T-cell subsets, and their inflammatory profiles will be defined in heart, blood, spleen, and mediastinal lymph nodes by flow cytometry and immunohistochemistry at serial time points after ligation, and correlated with LV and splenic remodeling and inflammation. BrdU labeling will index Treg proliferation in vivo. Moreover, using human HF biosample repositories, we will determine the abundance and distribution of Tregs in human failing hearts, and profile circulating Tregs (and other T-cells) in ambulatory patients with HF (with reduced ejection fraction) versus matched non-failing controls. In Aim 2, we will establish the role of Tregs in LV remodeling and neovascularization by selectively (and reversibly) depleting Tregs in Foxp3-DTR Tg mice with chronic HF, and then evaluating the effects on immune cell profiles, inflammation, circulating and bone marrow endothelial progenitor cells, in vivo angiogenesis, and LV remodeling. In parallel studies, the relative anti-angiogenic effects of splenic Tregs from sham mice, HF mice, and HF mice with ablation and subsequent reconstitution of Tregs will be compared using in vitro tube formation assays. In Aim 3, we will define whether dysfunctional Tregs are important mediators of the injurious cardiosplenic axis in chronic HF. CD45.2 mononuclear splenocytes from Foxp3-DTR Tg mice with either early or late HF, (corresponding to different stages of Treg dysfunction) will be adoptively transferred into CD45.1 nave mice, with or without Treg ablation prior to transfer. The long-term effects of cell transfer on LV/splenicremodeling, inflammation, and T-cell/immune cell profiles will then be measured in recipient mice. In parallel, we will evaluate the ability of splenic Tregs from HF and sham mice to suppress activation of CD4+ and CD8+ splenic T-lymphocytes in vitro, and the role of TNFR signaling in HF Treg dysfunction via similar studies using splenic T-cells from TNFR1-/- and TNFR2-/- HF mice. By conclusively defining the role of Tregs in pathological cardiac remodeling, these studies will further our understanding of the cellular basis for inflammatory activation in chronic ischemic HF, and provide innovative perspectives as to the fundamental underpinnings of the recently discovered pro-inflammatory and tissue-injurious cardiosplenic axis. Moreover, by providing direct evidence for Tregs as anti-angiogenic and (paradoxically) pro-inflammatory mediators in HF, these studies will identify new targets for cell type-specific, rather than cytokine-specific, immunomodulation.